1. What is the turnaround time?
The entire RPPA process takes approximately 8-10 weeks to complete (This time frame accounts for sample accumulation and sample processing time). Once we accumulate 1,000 samples from customers to accommodate a full set, the RPPA processing time is approximately 4 weeks. Service is provided on a "first-come, first-served" basis. We encourage you to submit your online forms and samples as soon as possible.
*Note: You cannot submit your samples until your forms have been fully completed and approved, and you are notified of approval to submit your samples*
2. How should I prepare my samples? Do you have special protocols?
Yes, we have protein extraction protocols posted under the Antibody Information & Protocols Page. We can also provide lysis buffer and SDS sample buffer for your sample preparation. Please contact the Core to arrange a pick-up or shipment of the buffers. If we will be shipping the buffers, a valid FedEx number for shipping charges will be required.
3. After completion, what information and data can I expect to receive from the Core?
Your data set will be arranged in an Excel file. It includes raw data in log2 value and normalized data in linear value. You will be able to generate bar graphs or other formatted graphs using normalized linear value. We also provide brief heatmaps with median-centered log 2 values. These heatmaps represent visualization figures for your reference. Please consult your bioinformatics group for further detailed analysis.
4. How do you normalize the data set?
We normalize each dataset by protein loading. Unlike western blots by b-Actin or GAPDH, we use the entire panel of antibodies to normalize protein loading. The normalization process is:
1. Convert raw data from log 2 value to linear value.
2. Determine median for each antibody across the sample set.
3. Divide each raw linear value by the median within each antibody to get the median-centered ratio.
4. Calculate the median from median-centered ratio (from Step 3) for each sample across the entire panel of antibodies. This median functions as a correction factor (CF.1) for protein loading adjustment, and each sample has its own correction factor. We consider the sample an outlier if the correction factor is above 2.5 or below 0.25.
5. Divide each median-centered ratio in linear value (from Step 3) by the correction factor (CF.1 from Step 4) to get the normalized linear value.
We calculate the correction factor and provide you the normalized linear value. We also list the correction factor (CF.1) in the Excel file (the last column on the page of normalized linear value) for your reference to determine sample outliers.
5. How do we quantify protein expression and modification?
We use the approach of "Supercurve Fitting" developed by the Department of Bioinformatics and Computational Biology at MD Anderson Cancer Center to quantify protein expression and modification. Briefly, a "standard curve" is constructed from 5808 spots on each slide (one slide probed for one antibody). These spots include 5 serial dilutions of each sample plus 528 QC spots of standard lysates at different concentrations. Relative levels of protein expression and modification for each sample are determined by interpolation of each dilution curve to the "standard curve" (supercurve) of the slide (antibody).
6. If I have several RPPA data sets that were performed at different times within different RPPA assays, can I combine all my data together for further analysis?
As with any other biological assays, there are batch variations between each RPPA assay. At this time, it is not possible to combine the raw or protein loading normalized values. We are working with The Department of Computational Sciences and Bioinformatics to develop an approach for data merging. However, in any case, you should design constant internal controls that will be included within each experiment, to be used for data merging in future RPPA assays.
7. I have samples ready but they were prepared in the buffer for my western blot. Can I submit these samples for RPPA?
We encourage you to use the lysis buffer with our recipe. However, we accept samples prepared by the approach for western blot. Our experience demonstrates that samples prepared in RIPA buffer will work for RPPA. However, samples prepared from Urea buffer will make diffused spots that interfere with quantification.
8. What is the optimal protein concentration of the samples for RPPA? If my sample concentration is below 1 ug/ul, can you run this sample for RPPA?
The optimal protein concentration should be 1 ug/ul after SDS denature. We accept samples with a minimal concentration of 0.50 ug/ul (from cell lines), and 0.75 ug/ul (from tissue). If you have few samples with low protein concentration in your sample set, we suggest you not to adjust protein concentration against the lowest one. Otherwise, you will lose the majority of the signals.
9. How much volume of each sample do you need for RPPA?
We require a minimum 80 ul of each sample to accommodate the entire Standard Panel of Antibodies.
10. Do I need to prepare replicate samples for RPPA?
There are two kinds of replicates: biological replicate and technical replicate. Please design replicates according to your study. We do charge replicates as separate samples. We perform multiple technical replicates using our standard lysates across the slides on different positions. These replicates will function as quality control for each antibody in RPPA. We release the result from those antibodies which pass our quality control, but it is up to your discretion if and how many replicates you choose to submit, although we encourage biological replicates, for technical replicates hold no real benefit.
11. Do I need to select antibodies for RPPA?
No, we have a Standard Panel of Antibodies that we use for each set. Please refer to the Antibody Information & Protocols Page. You do not need to select antibodies from the Standard Panel, we will perform the entire panel of antibodies on this list and release the results from the antibodies that passed our quality control.
12. If the antibody against the protein of my interest does not exist in your list, can I provide you with an antibody?
Yes, you can provide your own antibodies of interest. Since we do not have experience using your antibodies, it may take several runs to get the optimized dilution for RPPA. You will need to provide 50ul of the antibody along with the data sheet and Western Blot results (if applicable).We charge $750 per sample set for each supplied antibody that has never been run by the RPPA Core. We charge $300 per sample set for each supplied antibody that has been previously run on a RPPA Set. Please note that each antibody in our list is validated for RPPA application. The antibodies you submit have not been validated, thus, we can not guarantee the result.
13. If I would like you to validate my antibody, do you provide such a service?
Yes, we charge $750 per antibody. To request antibody validation, please submit a set of samples together with your western blot result with your antibodies and densitometry measurements on each band. We will perform RPPA on your samples. Please provide your antibodies together with the source information, such as host-rabbit or mouse, company and catalog number, etc. The validation will be judged on the following criteria: (A) Single band on western blot with right molecular size, (B) Acceptable dynamic range on the specific protein manipulated by chemical or genomic approaches, (C) Good correlation between western blot and RPPA (r > 0.7),(D) Acceptable Signal/Noise ratio on RPPA.
14. I have a set of tissue samples to be submitted for RPPA analysis. How much tissue do you need from each sample? Do you have protocols to extract proteins from tissue?
Yes, our experience demonstrates that approximately 10 mg of frozen tissue (approximately the size of a grain of rice) for each sample is required to process RPPA for the entire panel of antibodies. We prefer to have your tissue samples prepared in our lysis buffer.
15. Do you provide service to extract protein from tissue or cell pellets for RPPA?
Yes. We provide protein extraction from fresh frozen tissue and dry frozen cell pellets. We do not accept FFPE samples at this time. We require about 10mg of tissue (about the size of a grain of rice), or 5 millions cells (1 million minimum).
16. I have a set of clinical samples. Can I submit them for RPPA analysis?
Yes. You must delete patient sensitive information, such as Medical Record Number, Name, etc., before you submit. We will not accept samples with patient sensitive information.
17. I am in the process of submitting a grant application and/or manuscript which will involve RPPA analysis. Do you have any information I can refer to in my grant application?
Yes. Please refer to the Education and References page.
18. I am a research investigator outside of MD Anderson Cancer Center. How should I pay for the RPPA service?
You will need to complete the online submission process through iLab Solutions and include a valid purchase order number or complete the credit card authorization form. Full payment is due no later than 30 days after receipt of invoice.
19. Do I need to validate my result from RPPA?
Yes. RPPA provides you a result of cell signaling networks in a kind of "screen" mode. You need to validate and confirm your result by other technical approaches.